The primary objective of this proposal is to identify cellular genes that regulate hepatitis C virus (HCV) replication in the hepatocyte. The application is based on recently described liver gene expression profiles that characterize different time points during the course of HCV infection in experimentally infected chimpanzees. Three groups of genes were identified whose expression correlated with (a) the onset and duration of infection, (b) the magnitude of infection, and (c) the resolution of infection. Several genes involved in lipid metabolism were shown to be associated with the magnitude of infection in those studies. Experiments designed to validate the relevance of those observations revealed that replication of a subgenomic HCV replicon in Huh-7 cells was enhanced or suppressed by drugs that stimulate or inhibit cholesterol and fatty acid biosynthesis, respectively. In the current application, Huh-7 cells containing subgenomic and full length HCV-replicons will be used to determine if any of the genes identified in the infected chimpanzees can control HCV replication in hepatocytes. Expression of these genes in Huh-7 cells will be inhibited by RNA interference or enhanced by transfection, and the effect of those manipulations on HCV replication will be assessed. Specific Aims 1-3 will examine whether the three groups of liver genes identified in the chimpanzees are required either to maintain basal levels of HCV replication (Aim 1), to enhance HCV replication above basal levels (Aim 2); or to mediate the antiviral effects of interferon (Aim 3). In addition, in Aim 4, the specific step(s) in cellular cholesterol and fatty acid biosynthesis that regulate HCV replication will be identified, and the impact of cellular lipid metabolism on the intracellular localization and interactions of HCV proteins will be assessed. By elucidating the cellular regulatory mechanisms that control HCV replication, the studies described in this application may identify new targets for therapeutic antiviral intervention.